Palladin interacts with SH3 domains of SPIN90 and Src and is required for Src-induced cytoskeletal remodeling.

Palladin and SPIN90 are widely expressed proteins, which participate in modulation of actin cytoskeleton by binding to a variety of scaffold and signaling molecules. Cytoskeletal reorganization can be induced by activation of signaling pathways, including the PDGF receptor and Src tyrosine kinase pathways. In this study we have analyzed the interplay between palladin, SPIN90 and Src and characterized the role of palladin and SPIN90 in PDGF and Src-induced cytoskeletal remodeling. We show that the SH3 domains of SPIN90 and Src directly bind palladin's poly-proline sequence and the interaction controls intracellular targeting of SPIN90. In PDGF-treated cells, palladin and SPIN90 co-localize in actin-rich membrane ruffles and lamellipodia. The effect of PDGF on the cytoskeleton is at least partly mediated by the Src kinase since PP2, a selective Src kinase family inhibitor, blocked PDGF-induced changes. Furthermore, expression of active Src kinase resulted in coordinated translocation of both palladin and SPIN90 to membrane protrusions. Knock-down of endogenous SPIN90 did not inhibit Src-induced cytoskeletal rearrangement, whereas knock-down of palladin resulted in cytoskeletal disorganization and inhibition of remodeling. Further studies showed that palladin is tyrosine phosphorylated in cells expressing active Src indicating bidirectional interplay between palladin and Src. These results may have implications in understanding the invasive and metastatic phenotype of neoplastic cells induced by Src.

The Z-disc proteins myotilin and FATZ-1 interact with each other and are connected to the sarcolemma via muscle-specific filamins.

Myotilin and the calsarcin family member FATZ-1 (also called calsarcin-2 or myozenin-1) are recently discovered sarcomeric proteins implicated in the assembly and stabilization of the Z-discs in skeletal muscle. The essential role of myotilin in skeletal muscle is attested by the observation that certain forms of myofibrillar myopathy and limb girdle muscular dystrophy are caused by mutations in the human myotilin gene. Here we show by transfection, biochemical and/or yeast two-hybrid assay that: (1) myotilin is able to interact with the C-terminal region of FATZ-1 and that the N- or C-terminal truncations of myotilin abrogate binding; (2) myotilin can also interact with another calsarcin member, FATZ-2 (calsarcin-1, myozenin-2); (3) myotilin and FATZ-1 bind not only to the C-terminal region of filamin-C containing the Ig repeats 19-24, but also to the other two filamins, filamin-A and filamin-B, as well as the newly identified filamin-Bvar-1variant; (4) the binding of myotilin to filamin-C involves binding sites in its N-terminal region, whereas FATZ-1 associates with filamin-C via sequences within either its N- or C-terminal region; and finally, (5) the C-terminal region of filamin-C like filamin-B and filamin-Bvar-1, shows binding activity with the beta1A integrin subunit. Our findings further dissect the molecular interactions within the Z-disc that are essential for its organization, and provide evidence for a novel connection between Z-disc proteins and the sarcolemma via filamins and beta1 integrins. These data shed new light on the complex organization of the Z-disc that is highly relevant to understanding muscular dystrophies.

Involvement of palladin and alpha-actinin in targeting of the Abl/Arg kinase adaptor ArgBP2 to the actin cytoskeleton.

Palladin and alpha-actinin are major components of stress fiber dense bodies, cardiomyocyte Z-discs and neuronal synapses. They function as structural molecules and cytoskeletal regulators but also as docking sites to other proteins. Both antisense and transient overexpression experiments have shown that palladin plays an important role in the regulation of actin cytoskeleton. ArgBP2 is a multi-domain scaffolding protein which shares both the tissue distribution and subcellular localization with palladin. ArgBP2 is directly linked to intracellular signaling cascades by its interaction with Abl family kinases, Pyk2 and the ubiquitin ligase Cbl. It has several actin associated binding partners and has been shown to regulate cytoskeletal dynamics. Here, we show by in vivo and in vitro methods that palladin's amino-terminal poly-proline sequences directly interact with the first carboxy-terminal SH3 domain of ArgBP2. We further demonstrate a direct interaction between alpha-actinin and the amino-terminal segment of ArgBP2. Immunoprecipitation and targeting assays suggest that a three-way complex of the proteins occurs in vivo. The interactions provide an explanation to the previously observed Z-disc-specific localization of ArgBP2 and indicate interplay between signaling adaptors and structural proteins of the Z-disc.

Molecular analysis of the interaction between palladin and alpha-actinin.

Palladin is a novel component of stress fiber dense regions. Antisense and transient overexpression studies have indicated an important role for palladin in the regulation of actin cytoskeleton. Palladin colocalizes and coimmunoprecipitates with alpha-actinin, a dense region component, but the molecular details and functional significance of the interaction have not been studied. We show here a direct association between the two proteins and have mapped the binding site within a short sequence of palladin and in the carboxy-terminal calmodulin domain of alpha-actinin. Using transfection-based targeting assays, we show that palladin is involved in targeting of alpha-actinin to specific subcellular foci indicating a functional interplay between the two actin-associated proteins.

Myotilin, the limb-girdle muscular dystrophy 1A (LGMD1A) protein, cross-links actin filaments and controls sarcomere assembly.

The assembly and maintenance of the muscle sarcomere requires a complex interplay of actin- and myosin-associated proteins. Myotilin is a thin filament-associated Z-disc protein that consists of two Ig-domains flanked by a unique serine-rich amino-terminus and a short carboxy-terminal tail. It binds to alpha-actinin and filamin c and is mutated in limb girdle muscular dystrophy 1A (LGMD1A). Here we show that myotilin also directly binds F-actin, efficiently cross-links actin filaments alone or in concert with alpha-actinin and prevents filament disassembly induced by Latrunculin A. Myotilin forms dimers via its carboxy-terminal half, which may be necessary for the actin-bundling activity. Overexpression of full-length myotilin but not the carboxy-terminal half induces formation of thick actin cables in non-muscle cells devoid of endogenous myotilin. The expression of myotilin in muscle cells is tightly regulated to the later stages of in vitro myofibrillogenesis, when preassembled myofibrils begin to align. Expression of either amino- or carboxy-terminally truncated myotilin fragments but not wild-type myotilin in differentiating myocytes leads to myofibril disarray. The disease association and functional characteristics indicate an indispensable role for myotilin in stabilization and anchorage of thin filaments, which may be a prerequisite for correct Z-disc organization.